The protein .beta.-amyloid (A4, A.beta., A.beta..sub.1-39-42) has long been central to the neuropathology of Alzheimer's disease [Glenner and Wong, 1984]. However, its role in the disease process of Alzheimer's disease and other diseases, as well as its mechanism of action, remains in dispute.
It is undisputed that .beta.-amyloid protein is a major component of the neuritic plaques which, along with the neurofibrillary tangles, provide the neuropathological diagnostic markers for Alzheimer's disease [Mattson, 1995; Vantner et al., 1991]. It is deposited around cerebral blood vessels in Alzheimer's disease [Scholz, 1938; Mandybur, 1975; Vinters, 1987].
The sequence for .beta.-amyloid is known [Glenner et al. 1984].
Emphasis has been on Alzheimer's being a neurological disease, not a vascular disease.
It has been suggested in Alzheimer's disease pathogenesis that .beta.-amyloid has putative neurotoxic properties. However, there has been no consistent detection of such neurotoxic effects and there are conflicting reports [Price et al. 1992].
Referring to Teller et al. (1996), deposits of insoluble fibrils of amyloid .beta.-peptide (A.beta.) in the brain is is a prominent neuropathological feature of all forms of Alzheimer's Disease (AD) regardless of the genetic predisposition of the subject. In addition to the deposition of A.beta. in senile plaques and neurofibrillary tangles, vascular amyloid deposition resulting in cerebral amyloid angiopathy is a hallmark of AD and related disorders such as Down's Syndrome. The abnormal accumulation of A.beta. is due to either over expression or altered processing of amyloid precursor protein (APP), a transmembrane glycoprotein. Soluble A.beta. containing forty amino acids (A.beta..sub.40) and to a lesser degree the peptide with forty-two amino acids (A.beta..sub.42) forms the core of the amyloid deposits. The APP gene is highly conserved across different species and APPmRNA has been detected in all tissues, suggesting a normal physiologic role for A.beta.. The cellular origin of A.beta. deposited in the brain or cerebral blood vessels in AD or its precise role in the neurodegenerative process has not been established.
Another prominent etiologic theory of Alzheimer's disease recognizes and attempts to make consistent the etiology of the disease with the free radical theory of aging [Harman, 1986]. Free radicals are known to be neurotoxic and have been implicated with Alzheimer's disease pathophysiology. However, there has been no direct relationship shown or even suggested between .beta.-amyloid and free radical formation. Again, these theories revolve around the concept of a neurological disease and not any contribution of a vascular disease.
The generation of toxic oxygen radicals by A.beta. has been investigated as a factor contributing to the neurotoxicity of A.beta. and the observation that Apolipoprotein .epsilon.4 allele is a risk factor for both vascular diseases and AD has renewed interest in the vascular abnormalities in AD. Applicants have discovered a novel and direct action of A.beta. on blood vessels [Thomas et al. (1996) in press]. Applicants have demonstrated that A.beta. interacts with endothelial cells on blood vessels causing an imbalance in the ratio of nitric oxide to other free radicals, notably superoxide, with attendant endothelial dysfunction and/or damage and alterations in vascular tone. Free radicals are known to mediate normal physiology of vasculature and modest disruption of this system would result in dysfunction but not necessarily damage. In normal situations, levels of antioxidants may be adequate to provide protection.
The present invention recognizes, for the first time, an association between .beta.-amyloid peptide-induced free radical excess and resultant small vessel diseases including Alzheimer's disease. Although large blood vessel disease with dementia is considered distinct from Alzheimer's disease, several studies note similar pathological changes in the microvasculature in Alzheimer's disease and related neurodegenerative disorders [Buee et al., 1994].
The present invention recognizes the vasoactive effect of .beta.-amyloid peptides, its relationship to the production of free radicals, and, therefore, recognizes a direct association between .beta.-amyloid and vascular function and vascular degenerative diseases. The discovery further suggests the possibility that .beta.-amyloid activity at the endothelial cell surface can precipitate damage to neurons and result in Alzheimer pathology. Based on these discoveries, therapeutic and diagnostic protocols have been developed to utilize these properties.
The present invention further recognizes the effects of different lengths of solubilized .beta.-amyloid peptides in human aortic endothelial cell lines. Mutations in the .beta.-amyloid precursor protein (.beta.-APP) gene lead to early onset AD. An allelic variant of .beta.-APP, causes Hereditary Cerebral Hemorrhage with amyloidosis, Dutch type (HCHWA-D) which is characterized by severe cerebral amyloid antipathy (CAA) and hemorrhage.
Applicants further investigated the possibility that .beta.-amyloid peptides induce the release or enhance the activity of endogenous vasoconstrictors. Accordingly, the present invention relates to the compared effects of .beta.-amyloid peptides on the constriction evoked by other known vasoconstrictors, such angiotensin II and endothelin-I. Since enhancement of contraction of this system was always significantly increased in the presence of A.beta..sub.1-40, applicants employed this to reexamine .beta.-amyloid peptide enhancement and the effects of SOD to compare vasoactivity of different .beta.-amyloid peptide fragments.
Recent data suggest that production of the A.beta..sub.1-40 peptide may be the common factor in the pathology from .beta.-APP, PS-2 mutations to AD pathology (Scheuner et al., 1996), applicants compared the potency of A.beta..sub.1-40 and A.beta..sub.1-42 to enhance ET-1 induced vasoconstriction. Applicants also examined the vasoactivity of A.beta..sub.25-35 which is widely regarded to be the neurotoxic fragment of AB (Behl, et al., 1994). In addition, to clarify the mechanism of A.beta. vasoactivity applicants used both radioimmunoassay and pretreatment with allopurinol to determine if endogenous ET-1 was being released by A.beta. addition. To investigate the site of action of AB applicants compared enhancement in the presence and absence of endothelium, and also observed enhancement in the presence of specific ET-1 receptor antagonists.